Exploration of host cells during old world alphavirus infection : modulation of RNA granules and the PI3K/AKT pathway

Abstract: As obligate intracellular parasites, viruses have to explore and modulate cellular pathways for their survival. Mechanistic studies of virus–host interactions provide a better understanding of viral infection and cellular responses and help to identify potential targets for therapeutic intervention. Alphaviruses, a group of small enveloped viruses with positive-sense, single-stranded RNA genomes, are transmitted by mosquitoes and pose threats to human health. Semliki Forest virus (SFV) belongs to this group and has been extensively studied as a model virus for alphaviruses. Infection with alphaviruses, such as the important human pathogens chikungunya virus (CHIKV) or Ross River virus (RRV), is characterized by high fever, rash and debilitating joint pain, which can last for months or even years. The recent outbreaks and expanding spread of CHIKV in many tropical regions of the world as well as the continuous endemic of RRV in Australasia highlighted the significance of alphavirus research. Stress granules (SGs) are cytoplasmic aggregates of non-translated messenger ribonucleoprotein particles (mRNPs) that can be induced by many types of environmental stress, including viral infection. RNA processing bodies (P-bodies) are cytoplasmic aggregates of translationally silent mRNA and proteins involved in mRNA decay and translation repression. Unlike SGs, P-bodies are constitutively present in the cytoplasm under normal conditions. Both granules can actively respond to environmental stress and associate with each other under certain conditions. The PI3K–Akt–mTOR pathway plays important roles in regulating the transition between cell anabolism and catabolism, responding to changes in the cellular environment. Proper responses of SGs, P-bodies or the PI3K–Akt–mTOR pathway are seen as important adaption for cell survival under stress conditions. In this thesis, it was investigated how alphaviruses (more specifically, Old World alphaviruses such as SFV, CHIKV and RRV) exploit the SG nucleating protein G3BP, P-bodies and the PI3K–Akt–mTOR pathway upon infection. Previous studies have demonstrated that the alphavirus non-structural protein nsP3 binds to the SG nucleating protein G3BP via its two FGDF motifs to block SG induction. In paper I, we compared the two FGDF motifs in nsP3 with respect to their contribution to G3BP binding. The three-dimensional structure of G3BP1 bound to an SFV nsP3-derived peptide (nsP3-25, containing two FGDF motifs) revealed a poly-assembly of G3BP1 dimers inter-connected by nsP3-25. Both in vitro and in vivo binding studies demonstrated a hierarchical binding mode of the duplicate FGDF motifs to G3BP. SFV mutants lacking either of the FGDF motifs failed to bind levels of G3BP necessary for efficient replication, clearly demonstrating that both intact FGDF motifs are required for efficient virus replication. The hierarchical binding mode of two FGDF motifs was also observed for CHIKV nsP3. Growth curves showed that the two intact FGDF motifs are critical for viral replication. Mutation of both FGDF motifs was lethal to CHIKV. These results highlight a conserved molecular mechanism of virus-induced G3BP modulation. In paper II, we described that P-bodies are disassembled or reduced in number after infection with SFV or CHIKV in various cell lines. Disassembly of P-bodies occurs at an early stage (3–4h) post infection and is independent of viral structural protein expression. Similar to SGs, P-bodies could not be re-introduced by a second stress (sodium arsenite) in infected cells. However, formation of SGs or communication between P-bodies and SGs is not necessary for P-body disassembly, since P-bodies were still disassembled to a similar extent upon infection of cells incapable of forming SGs. Studies of the translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of the eukaryotic initiation factor eIF2a in cells infected with SFV or CHIKV. By labelling newly synthesized RNA with bromo-UTP, we observed that the timing of host transcription shutoff correlated with P-body disassembly, occurring at the same stage (3–4h) after infection. However, inhibition of transcription with actinomycin D (ActD) failed to disassemble P-bodies as efficiently as the viruses did. Interestingly, the block of nuclear import in non-infected cells with importazole led to an efficient P-body loss. These results reveal that P-bodies are disassembled independently of SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamics of P-bodies. Infection with SFV hyperactivates the PI3K–Akt–mTOR pathway. In paper III, we show that a tyrosine residue in the sequence context YEPM in nsP3 of SFV is pivotal for this phenotype. When cells were infected with SFV carrying mutations that affect this motif, such as the replacement of the YEPM tyrosine by phenylalanine (SFV-YF), Akt activation was significantly reduced and delayed in comparison to wildtype SFV infection. Ectopically expressed nsP3 of SFV-WT but not SFV-YF activated the pathway, but only when provided with a membrane anchor. Co-immunoprecipitation experiments revealed that nsP3 of SFV-WT, but not SFV-YF, bound to the regulatory subunit p85 of PI3K, dependent on both the tyrosine of nsP3 and the SH2 domains of p85, which specifically interact with phosphorylated tyrosines in YXXM motifs. This indicates tyrosine phosphorylation of SFV nsP3. A similar YETM motif was identified and characterized in RRV nsP3. Similar to SFV, RRV-WT hyperactivated the PI3K–Akt–mTOR pathway while the mutant RRV-YF, in which the YXXM tyrosine in nsP3 was replaced by phenylalanine, failed to do so.. Complementary experiments showed that infection of cells with SFV or RRV reprograms cellular metabolism and that mutation of the critical tyrosine residue attenuates the virus in cell culture and, in case of RRV, also in mice. These results reveal that the hyperactivation of the PI3K–Akt–mTOR pathway by SFV and RRV, mediated by the interaction of nsP3 and p85, contributes to virus pathogenicity. Taken together, the data from papers I, II and III highlight and characterize three mechanisms by which Old World alphaviruses subvert cells – interactions with the SG nucleating protein G3BP, P-bodies and the PI3K–Akt–mTOR pathway, respectively. These viral subversion mechanisms are largely independent of each other and represent three different modes by which Old World alphaviruses ensure efficient virus growth.